Surface layer affinity-chromatography

ABSTRACT

There is described an affinity-chromatography assay system comprising with an immobilised component containing a bio-reagent and a flowable component containing a complimentary bio-reagent characterised in that the immobilised component is supported on a dip strip or planar surface and the flowable component is adapted to flow down the dip strip of high density. There is also described a method of conducting an affinity-chromatography assay which comprises the use of such an assay system.

Immuno-chromatography is currently performed in two major formats. Thefirst is performed within gels when development is achieved by passivediffusion or is electrochemically induced, and the second is performedin flow. In the former using gels, radial immunodiffusion is the mostcommonly used system with a preformed gel often located within acircular plate in which a central well is present in the gel togetherwith additional wells located around the edge of the gel. Antiserum orantigen is placed in the central well and antigen or antiserum is placedin the peripheral wells. Passive diffusion occurs within the gel andwhite bands of immunocomplex are seen within the gel due toantibody-antigen complex formation. In the electrochemical systemantibody/antigen migration within the gel is induced using an electriccurrent.

In the flow-based systems the antibody or antigen is often immobilisedwithin a cartridge which is paced with a liquid flow system such as aflow injection analysis system. The complementary antigen or antibody isinjected and flows through the cartridge where specific interactionstake place. Often the component that is injected carries a label thatcan be detected downstream, thereby producing a signal. Alternativelyflow occurs over a planar surface as in lateral flow diffusionimmunoassay systems where flow is induced by membranewetting/capillarity.

In the new format the equivalent immunoreactions take place in flowbetween an immobilised component and one in solution as above but thisnow occurs on the surface of a dip strip residing within a buffersolution. In this case flow across the surface of the strip occurs dueto the higher density of a solution containing one of the immunoreagentsthat is initially present at the top of the strip that is itselfstanding in the buffer solution. Since the strip is nearly upright thisdenser solution slowly rolls down the surface of the strip presentingthe reagent in the flowing phase to the immobilised reagent on thesurface of the dip strip.

Thus according to the invention we provide an affinity-chromatographyassay system comprising with an immobilised component containing abio-reagent and a flowable component containing a complimentarybio-reagent characterised in that the immobilised component is supportedon a dip strip or other planar surface and the flowable component ofhigh density is adapted to flow down the dip strip.

In order for this phenomenon to work certain criteria must be met.Firstly the denser solution must be retained in a discrete volume as itpasses as a layer over the surface rather than rapidly diffusing intothe bulk of the buffer solution. Secondly the dip strip must possesscertain properties that result in attraction of the rolling surfacelayer again leading to retention of the integrity of this mobile phase.To achieve the first criterion we have carefully chosen the constituentsof the rolling phase to include a polymeric agent such as a proteinand/or a polysaccharide, a detergent and a buffer of optimal pH, and forthe second we use a membrane that is both hydrophobic and wettable.

The system can be used as an immuno-chromatography system and assaysperformed in either competitive or non-competitive immunoassay formats.In the former the immobilised spot is either antibody or antigen. Forimmobilised antibody, a labelled antigen is deposited in a band abovethe spot in a cellulose square. A drop of sample containing the antigenis added to this square and after a suitable interval (1-10 min) thewhole strip is immersed in a buffer solution. The dense mixture oflabelled and unlabelled antigen flows over the spot of antibody andcompetition for binding takes place. If the antigen is immobilised atthe spot, a labelled antibody replaces the labelled antigen in thecellulose square and the assay proceeds as before.

The system can also be performed in a non-competitive immunoassay formatwhen a spot of capture antibody is immobilised on the strip. Labelledantibody is deposited on the cellulose square above the first spot. Thestrip is placed in the sample solution containing the antigen so thatthe upper square is immersed. Incubation now takes place during whichantigen in the solution is captured by immobilised antibody on the spot.At the same time the labelled antibody is reconstituted in a densesolution that flows over the spot after about 5-10 minutes followinginsertion of the strip into the sample solution. This time lag enablesantigen molecules to be captured on the spot prior to arrival of thesurface layer containing the labelled antibody. This second antibodylabels the captured antigen on the spot's surface.

In addition any label can be used. If a fluorescent or coloured label isused with the antibody or antigen, then a fluorescent or coloured spotwill result following the first incubation, making the assay a singlestep system.

If an enzyme label is used then a modified sequence of steps can be usedin the non-competitive assay. In this, enzyme-labelled antibody is nowadded to the cellulose square attached to the bottom of the stripbeneath the spot of immobilised capture antibody. A second cellulosesquare is also attached as before above this spot but this contains adried solution of substrate for the enzyme plus a biopolymer such asdextran. The strip is placed in a limited volume of sample as before soas not to wet the upper cellulose square. The dense reconstitutedsolution of labelled antibody flows to the bottom of the container andstays there as a separate layer. At the same time antigen in solution iscaptured by the antibody on the spot. At the end of this incubation step(5-10 minutes) the solution is stirred using the dip strip. This causesthe labelled antibody to be homogeneously distributed within thesolution and the antibody can now bind to the captured antigen on thespot. Finally the volume in the container is increased to wet the uppercellulose square. The substrate is now reconstituted as a dense solutionthat flows over the spot when substrate to product conversion takesplace resulting in a coloured spot.

According to a further aspect of the invention we provide a method ofconducting immuno-chromatography assays which comprise the use of anassay system as hereinbefore described.

This new surface layer chromatography phenomenon could, in theory, alsobe used as a generic chromatographic method for separation of analytemixtures if the components have different binding affinities for thesurface. For example, it is well known that biological polymers such asproteins and DNA/RNA bind to cellulose nitrate as this is used in DNA-and protein-blotting following electrophoresis. We have also observedthat for antibodies the rate of binding to this surface is pH sensitive[1]. Hence it should be possible to introduce a mixture of biologicalpolymers onto a cellulose strip above a cellulose nitrate square. The pHand density of the buffer used would be such that surface layerchromatography will ensue when the strip is immersed into a secondbuffer solution chosen to optimise the binding of the biopolymers to thesurface. Under these conditions, different binding interactions willtake place between the bio-molecules and the surface as the mobile phaselayer rolls over the surface. This should lead to separation of thecomponents during this development phase. The strip would then beremoved and the membrane treated to visualise, using establishedmethods, the now immobilised components of the mixture.

The invention will now be illustrated with reference to the accompanyingexample.

EXAMPLE 1

In an example of this new immunochromatographic system that we termSurface Layer Immuno-Chromatography (SLIC), a small square of cellulosenitrate membrane is pre-treated via established methods with a specificantibody to an antigen such as savinase, to produce a spot ofimmobilised reagent. This square is stuck on the surface of a plasticstrip pre-coated with one sticky surface. Above this is stuck a secondstrip of cellulose impregnated with a dried solution of the sameantibody labelled with the reporter enzyme alkaline phosphatase,together with bovine serum albumin (BSA) and Tween 20. The solution usedfor this deposition is Tris (pH 9.3) and a volume of 10 μl is used. Thecomposition of this solution is 0.1% w/v BSA, 0.1% w/v Tween 20,enzyme-labelled antibody diluted 1:1000 in Tris buffer (0.1M). Thereagents in this format when stored at room temperature in a desiccatorare stable on the strip for at least 14 days.

To perform the assay, a solution of savinase (0.3 ml) in Tris buffer isplaced in a test tube such as a conventional 96 well microtitre plate.The dip strip is now inserted into the well when both squares arecovered by the sample. On wetting, the reagents within the square ofcellulose pass into solution. Due to its higher density this solutionnow flows as a layer down the surface of the strip and eventually passesover the lower square containing the spot of immobilised antibody. Inthe time interval between immersion of the strip and arrival of theflowing phase, savinase molecules in the bulk solution will have beencaptured by the immobilised antibodies on the lower spot. On arrival ofthe flowing phase, labelled antibody will bind to the captured antigenmolecules as in a conventional sandwich-type ELISA, as the solutionflows over the spot. This first incubation period is typically 15minutes.

The strip is removed from the well and placed in wells containing thecommonly used substrate mixture for alkaline phosphatase,bromochloroindolyl phosphate (BCIP) and nitroblue tetrazolium salt(NBT). A purple/blue colour develops after 1 minute's incubation, theintensity of which is directly proportional to the amount of savinasecaptured on the spot during the first incubation step, and hence theconcentration of savinase in the sample. The top square is also colouredpurple/blue. A scan of the resulting strips is shown for this analyte.It should be noted that we use 12 such strips in a comb-like format asthis enables analysis of up to 96 samples/standards (8×12) to beperformed with a single microtitre plate. It will be noted that visualdiscrimination between the zero and the 5 ng/ml standard is clearlyseen.

EXAMPLE 2

As in example 1, a small square of cellulose nitrate membrane with aspot of specific antibody is attached to the surface of a plastic strip.Below this a second strip of cellulose (cut as 0.4×0.6 cm squares fromfast hardened filter paper) is placed. This square is impregnated with a5 μl of a diluted solution of the same antibody labelled with thereporter enzyme alkaline phosphatase, in a solution of blue dextran, (3%w/v), dextran (25 w/v), all in Tris buffer with azide, pH 9.3(containing 0.1% Tween 20 and 0.15 (w/v) bovine serum albumin). Theapplied solution is allowed to dry at ambient temperature for 30minutes. A third square of fast hardened filter paper is stuck above thefirst square and is impregnated with the substrate for alkalinephosphatase. This is prepared by addition of 15 μl of a mixture of 1%(w/v) blue dextran, 1% (w/v) dextran and BCIP-NBT stock solution. It isallowed to dry for 60 minutes at ambient temperature at which point thesquare is stuck to the dip strip.

The pre-prepared dip strips are then added to microcuvettes containing0.6 ml of standards or samples. After 10 minutes a blue layer ofsolution is observed at the base of the cuvette. The strips are used tostir the contents of the cuvettes. This produces a uniform bluecoloration throughout the solution. Incubate for a further 5 minutesthen add 500 μl of substrate buffer solution to now cover the top squareon the dip strip. Incubate for a final 10 minutes, then remove thestrips and wash with water.

When rabbit anti-savinase antibodies are used as the capture and cappingantibodies in the presence of savinase standards, the following stripsare observed when the above protocol is followed.

The resulting dip strips are illustrated in FIG. 1, in which:

A=zero standards, B=10 ng/ml standards of savinase

1. An affinity-chromatography assay system comprising with animmobilised component containing a bio-reagent and a flowable componentcontaining a complimentary bio-reagent characterised in that theimmobilised component is supported on a dip strip or planar surface andthe flowable component is adapted to flow down the dip strip of highdensity.
 2. An affinity-chromatography assay system according to claim 1characterised in that the flowable component is of a higher density thanthe bulk solution.
 3. An affinity-chromatography assay system accordingto claim 1 characterised in that immunoreagent is an antigen orantibody.
 4. An affinity-chromatography assay system according to claim1 characterised in that the flowable component is retained in a discretevolume.
 5. An affinity-chromatography assay system according to claim 1characterised in that the constituents of the flowable phase include abio-polymer, a detergent and a buffer of optimal pH
 6. Anaffinity-chromatography assay system according to claim 1 characterisedin that the immobilised component possesses properties that result inattraction of the flowable component.
 7. An affinity-chromatographyassay system according to claim 6 characterised in that the attractionof the flowable component is achieved by a membrane.
 8. Anaffinity-chromatography assay system according to claim 7 characterisedin that the membrane is both hydrophobic and wettable.
 9. Anaffinity-chromatography assay system according to claim 3 characterisedin that the assay is either a competitive or non-competitive immunoassayusing appropriate combinations of labelled antigen or labelled antibodywith their complementary unlabelled counterparts.
 10. Anaffinity-chromatography assay system according to claim 9 characterisedin that the label is a fluorescent or coloured label.
 11. A method ofconducting an affinity-chromatography assay which comprises the use ofan assay system according to claim
 1. 12. A method according to claim 11characterised in that the dipstrip that is stood substantially uprightin a buffer solution.
 13. A method according to claim 11 characterisedin that the flowable component is dispensed adjacent the upper or lowerpart of the dipstrip.
 14. A method according to claim 11 characterisedin that the method comprises the separation of analyte mixtures.
 15. Amethod according to claim 11 characterised in that the components havedifferent binding affinities for the surface.
 16. A method according toclaim 11 characterised in that the method comprises a single step assay.17. A method according to claim 11 characterised in that the methodcomprises the separation of biological polymers.
 18. A method accordingto claim 17 characterised in that the biological polymers are selectedfrom proteins and DNA/RNA.
 19. An affinity-chromatography assay systemor a method substantially as described with reference to theaccompanying examples.